Chapter 20 – Genomics and Gene Regulation



Chapter 20 Genomics and Gene Regulation

Jane Ding

1 Laboratory Techniques

1.1 Blotting

See Table 20.1.

Table 20.1 Blotting

Type Aim Process
Southern To detect the presence and amount of a particular DNA sequence in a sample with many DNA sequences

  1. 1. Electrophoresis: separates DNA fragments by size

  2. 2. DNA fragments blotted onto nitrocellulose membrane

  3. 3. DNA fragments fixed onto membrane

  4. 4. Membrane exposed to probe containing target DNA sequence

  5. 5. Target DNA sequence detection on X-ray

Northern To detect the presence and amount of a particular RNA sequence in a sample with many RNA sequences Similar to Southern blotting
Western To detect the presence and amount of a particular protein in a tissue sample

  1. 1. Electrophoresis: separates denatured proteins

  2. 2. Proteins fixed to membrane

  3. 3. Antibody (with radioactive tag) against target protein applied

  4. 4. Target protein detected on X-ray

1.2 Polymerase Chain Reaction (PCR)

  • aim: amplify specific DNA/RNA sequences in a sample with many DNA/RNA fragments

  • if RNA is being amplified, it must first be transcribed into complementary DNA

  • steps:

    1. 1. Attach primer onto target DNA

    2. 2. Taq polymerase adds nucleotides onto new DNA strand

    3. 3. Process repeated to produce many DNA copies

1.3 DNA Microarrays

  • aim: allows simultaneous analysis of thousands of gene expressions

  • microarray is a commercially produced collection of fluorescently labelled DNA short oligonucleotides

  • steps:

    1. 1. Isolate RNA from cells

    2. 2. Translate RNA into complementary DNA

    3. 3. Hybridise cDNA onto microarray oligonucleotides

1.4 Bioinformatics

  • definition: computing method to store, distribute and analyse large reported DNA information

  • researchers report DNA fragment information into databanks

  • databanks are accessible via the Internet

1.5 Proteomics

  • definition: the study of the structure and function of proteins

2 Cell Cycle Control: Cancer Development, Growth, Spread

2.1 Cancer

  • most cancers are caused by mutations in somatic cells (rather than germ cells)

  • it takes many years to accumulate the mutations (mutations need at least 20 years to cause cancer)

2.2 Cell Cycle Control Checkpoints

  • DNA damage checkpoints:

    1. where: S phase, G1, G2

    2. damage detected → cyclin-dependent kinase 2 (CDK2) inhibited → cell cycle progression stopped

    3. damage not repairable → apoptosis

  • spindle checkpoint:

    1. where: metaphase

    2. spindle fibres fail to attach to kinetochores → apoptosis

2.3 Signalling Proteins

  • signalling proteins include growth factors (+ receptors), signal transduction proteins and transcription factors

  • gain-of-function in these proteins can lead to cancer

  • mutated alleles usually dominant

2.4 Cell Cycle Control Proteins

  • cell cycle control proteins are usually tumour-suppressor proteins

  • loss of function in these proteins can lead to cancer

  • mutated alleles are usually recessive

  • adenomatous polyposis coli (APC):

    1. function:

      1. activates transcription factor Myc → transcribes genes pushing movement from G1 to S phase

    2. mutation:

      1. if APC mutated → inappropriately activates Myc → uncontrolled cell proliferation

      2. requires both APC alleles to be mutated for the protein to fail

  • p53:

    1. functions:

      1. detects DNA damage

      2. blocks CDK2

      3. activates apoptosis

    2. mutation:

      1. requires both p53 alleles to be mutated for the protein to fail

      2. fifty percent of cancers have mutations in p53

  • ataxia telangiectasia mutated:

    1. detects DNA damage

    2. stops cell cycle

    3. maintains normal telomere length: prevents chromosome shortening with DNA replication

2.5 Oncogenic Viruses

  • some viruses contain proto-oncogenes and oncogenes

  • DNA viruses:

    1. need oncogenes for their own viral survival

    2. example: human papillomavirus family

  • RNA viruses:

    1. retrovirus enters host → makes DNA copies from viral RNA → DNA copies inserted into host’s DNA for viral replication → oncogenes produced

    2. examples:

      1. Harvey sarcoma virus:

      1. contains Ha-ras gene (differs from the human ras gene by a single-point mutation)

      2. Ha-ras overexpression → bladder cancer

    3. Rous sarcoma virus

      1. produces v-Src protein, which is a constitutively active mutant of human c-Src protein

      2. leads to continuous phosphorylation of proteins

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Dec 29, 2020 | Posted by in GYNECOLOGY | Comments Off on Chapter 20 – Genomics and Gene Regulation

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